Many studies show a decreasing semen quality due to environmental and genetic causes. For example, it has been demonstrated that sperm counts are on the decline, and that other qualitative markers show high sensitivity to these effects, including DNA integrity. Due at least in part to incomplete maturation, cells generate free radicals (reactive oxygen species, ROS). ROS has been reported to cause DNA fragmentation and a loss of sperm function associated with oxidative damage to the sperm plasma membrane and the genetic material, in a sequence that depends on the source of such chemicals. Researchers are now learning that the quality of human sperm is steadily eroding, and might be causing not only infertility in men, but also childhood cancers in the offspring of those who can reproduce (Stewart Irvine, et al, 1996; Irvine D S, 2000; Irvine D S et al, 2000). For the men who can reproduce today, their sub clinically damaged sperm might lead to infertility in their male progeny (Hoyes K P et al, 1994). Continuous exposure to environmental toxins (pesticides, heavy metals, traffic pollutants, organic solvents and vapors, etc.) or electromagnetic radiation may impair sperm quality, affect the genetic integrity of the sperm DNA, or may alter the physiological structure of the sperm severely enough to interfere with the fertilization process (Moline J M et al, 2000; Massaad C et al, 2002). Cigarette smoking is also associated with an increased risk of infertility (Wong W Y et al, 2000; Kunzle R et al 2003). Sperm damage may also be caused by exposure of sperm to heat, such as soaking in hot tubs or hot baths (Karaca A G et al, 2002) or fever. Sperm genetic quality/nuclear integrity has been emphasized for several years as playing a role in early embryogenesis. T thus the rate of success of any assisted reproductive technique (ART) will be benefited where sperm are confirmed to have good DNA integrity.
Since the quality of sperm DNA is an important factor in fertilization and in the selection of spermatozoon for fertilizing an egg (as in intracytoplasmic sperm injection, [ICSI] during in vitro fertilization [IVF]), it is important to sperm quality more objectively, to focus on the assessment on genetic and DNA quality, and to assess late functions of the sperm cell. These factors remain relevant to ICSI procedures. Several methods and techniques have been developed to assess perm function and quality beyond the standard routine tests. The Sperm DNA Decondensation (SDD) Test is one part of a multi-event screening test named the Human Sperm Activation Assay (HSAA). The HSAA is used to examine sperm activation events (DNA decondensation, pronuclear formation, DNA synthesis, and DNA recondensation). The SDD Test is used to assess the process of sperm chromatin/DNA decondensation (Brown D et al, 1992, 1995). Sperm chromatin/DNA decondensation is the first sperm activation event that occurs after a spermatozoon penetrates an ovum (Brown D et al, 1992, Samocha-Bone D. et al, 1998), and is a factor to assess success rate in IVF-ET, ICSI, and other modes of ART. This event is a step in fertilization during which protamine disulphide bonds are reduced to SH, and in which the polycationic protamines combine with the polyanionic egg protein, nucleoplasmin, thus being stripped from DNA, which then combines with histones, allowing the formation of a pronucleus. As the DNA decondenses, the DNA is reformatted such that upon combining with the female's DNA during syngamy, and after the subsequent first cleavage that results in a 2-cell stage embryo, the developmental program is set in motion. A partially reformatted sperm nucleus will have the capacity to initially fertilize the egg, and even under go a number of cell divisions when used in IVF, ICSI, or other ART modes. However, the resulting embryo will not have the capacity to develop to term. Thus, it is important to have pre-ART tests performed to detect males producing sperm with compromised genomic integrity. Without such pre-testing, especially in the cases of IVF and ICSI attempts at pregnancy, the embryologist will often be providing the treating physician with embryos that will not likely develop to term after transfer.
Protamine defects in sperm (genetic or induced) are also correlated with male infertility. It is therefore suggested that the SDD Test provides a probe for the functional integrity of these specific steps in fertilization, and a better predictor of outcome of IVF, especially in the case of ICSI, as well as IUI's or natural inseminations. The test has been applied successfully on both human and rat sperm to detect damage by environmental toxicants, specifically, alkylating agents (Sawyer and Brown, 1995; Sawyer et al, 1998; Sawyer and Brown, 2000). Exposure of the sperm to alkylating agents results in a diminished decondensation. Recently, it has been shown that exposure of the sperm to oxidative stress (reactive oxygen species; ROS) can accelerate the decondensation process (Tirado et al, 2003; Tirado et al, 2004). Hence, both the presence or absence of decondensation must be examined, but likewise, the kinetics/dynamics of the morphological changes are informative.
The process of fertilization involves a series of events including: a) sperm binding to, and then penetration of the oocyte zona pellucida via the acrosome reaction, b) sperm fusion with the oocyte plasma membrane, c) oocyte activation (release of the cortical granules to prevent multiple sperm from fertilizing the egg), d) sperm activation and pronuclear formation (Abou-Haila A. et al. 2000: Furlong, L. et al. 2005).
Sperm activation initiates post-fertilization upon entry into the ooplasm where the compacted sperm DNA undergoes many alterations as it develops into a male pronuclear. During the first phase of sperm activation or decondensation, the chromatin condensation acquired during spermiogenesis is reversed. Protamines are exchanged with histones reformatting the sperm chromatin allowing for pronucleus formation (Ballhorn et al., Wasserman et al.). Once the sperm decondensation is complete, the DNA synthesis phase occurs, that is followed by recondensation of the sperm chromatin in preparation for the cell division that will result in the 2-cell stage embryo (Longo et. al., (1991)).
A need exists in the medical arts for a clinical male screening panel that includes assessment of early sperm activation events, preferably as part of a panel of tests that can be correlated and used in assessing male reproductive health. A need also continues to exist for a more comprehensive male reproductive health panel that may be used to prescribe an assisted reproductive technology (ART) tailored to the particular male patient or animal and/or reproductively challenged couple.